The long-term objective of this project is to understand the biological significance of the complex between ferric ions and non-amidated gastrins (NAGs). This laboratory has shown that NAGs selectively bind 2 ferric ions that ferric ion binding is essential for biological activity in vitro, and that gastrin interacts with transferrin. The specific aims of the project are: (1) to determine whether or not ferric ions are essential for the stimulation of colorectal carcinoma (CRC) development by NAGs in vivo, (2) to develop NAG antagonists, (3) to establish the role of NAGs in iron homeostasis, and (4) to define the role of the NAG-transferrin complex in cellular ion uptake and determine its structure. The health significance of the project lies in the facts that NAGs act as growth factors for the normal gastric and colonic mucosa, accelerate the development of both gastric and colorectal cancer, and may be involved in disorders of iron homeostasis. The research design mirrors the specific aims, and utilizes the unique combination of skills of the principal investigators. Firstly, agents known to block the binding of ferric ions to NAGs will be tested as NAG inhibitors in four animal models of CRC development. Secondly, exchange inert metal ion-NAG complexes and structurally modified gastrin fragments will be tested as NAG inhibitors in CRC cell lines and in animal CRC models. Thirdly, patterns of progastrin expression and processing will be measured by radioimmunoassy in mice with altered dietary iron uptake, and in patients with hemochromatosis. Iron status will also be measured in hypergastrinemic and gastrin-deficient transgenic mice with altered dietary iron uptake, and in patients with hypergastrinemia. Fourthly, the role of the gastrin/transferrin complex in cellular iron trafficking will be investigated, and covalent cross-linking and X-ray crystallography with a panel of NAG and transferrin mutants will be used to define the structural requirements for formation of the complex. These studies are expected to demonstrate an unexpected role for ferric ions in gastrin bioactivity in vivo, and for gastrins in ferric ion homeostasis. Recognition that metal ions are essential for the biological activity of NAGs may permit the development of novel therapies for colon cancer.